Study on the technology of brewing red raspberry wine by using new immobilized yeast technology

The aim of the present study was to establish a new process for brewing red raspberry wine with immobilized yeast and improve the value of red raspberry. Using the spore filaments of P. chrysogenum as a carrier to co-culture with yeast to obtain immobilized yeast. And the preparation method of immobilized yeast was optimized by single-factor experiment and orthogonal experiments. And the red raspberry wine was brewed in these conditions. The result shows that the optimal preparation conditions for the immobilized yeast of P. chrysogenum were 5 g/L gluconic acid, 5 g/L yeast extract, and the addition amount of P. chrysogenum spores 1 × 105 cfu/ml. Compared with free yeast, the immobilized yeast of P. chrysogenum has better fermenting ability, can better keep the anthocyanins and polyphenols and other active ingredients of red raspberry. The quality of the wine was improved due to increase in aroma components such as alcohols and esters. The immobilized yeast of P. chrysogenum maintained good fermentation performance after three consecutive fermentations.

Preparation method of immobilized yeast. Add an appropriate amount of sterile water to the PDA medium of Penicillium chrysogenum grown for 7 days, gently scrape the surface of Penicillium chrysogenum filaments with a spatula, pour the mixture into a sterile conical flask, and repeat the operation for 3 Second-rate. Shake the collected mycelial spore mixture, filter the mycelium with sterile gauze, and the obtained filtrate is the spore suspension of Penicillium chrysogenum. Yeast was added for co-cultivation, and the yeast was encapsulated by Penicillium chrysogenum to produce immobilized yeast. The immobilized yeast needs to be inactivated by Penicillium chrysogenum before use 12 .
Optimization of preparation conditions for immobilized yeast. Carbon source optimization: keep the basic immobilized yeast medium and culture method unchanged, use 3 g/L ammonium sulfate and 2 g/L yeast extract as nitrogen sources, and use glucose, sucrose, lactose, gluconic acid and glycerol as carbon sources respectively. The addition amount is 5 g/L. Nitrogen source optimization: other conditions remain unchanged, 5 g/L gluconic acid is used as carbon source, 2 g/L yeast extract is added, urea, ammonium sulfate, potassium nitrate, yeast extract and peptone are used as nitrogen sources, and the amount added is 3 g/L.
Optimization of P. chrysogenum spore addition: 5 g/L gluconic acid was used as carbon source, 5 g/L yeast extract was used as nitrogen source, and P. chrysogenum spore addition was 6 × 10 4 /mL and 1 × 10 5 /mL, respectively., 6 × 10 5 /mL, 1 × 10 6 /mL, 6 × 10 6 /mL. Physical property detection of immobilized yeast. Determination of the diameter of immobilized yeast: 10 mycelial balls were randomly selected and arranged in a straight line. Under the premise of maintaining the shape of the mycelial ball, an appropriate amount of filter paper was used to absorb the excess water on the surface of the mycelial ball. Measure the treated mycelium ball with a vernier caliper, and read the diameter value 11 .
Determination of the mechanical strength of immobilized yeast (texture analyzer method): The determination was carried out in the TPA mode of the texture analyzer, and the instrument probe was a universal cylindrical probe (diameter 45 mm; length 40 mm). The parameters of the TPA test program are set as the test speed of 100 mm/min, the deformation amount of 50%, the volume sensing element range of 25 N, the initial force of 0.04 N, and the pause time of 2 s. The measurement parameters are hardness and elasticity. Place the randomly selected immobilized yeast on the test bench. On the premise of maintaining the shape of the mycelial ball, use filter paper to absorb the excess water on the surface of the mycelial ball. After each test, wipe the probe clean before proceeding to the next test. Each test sample was measured 3 times, and the results were averaged.
Determination of the number of yeast encapsulated by immobilized yeast: 1 g of immobilized yeast was ground and crushed in a mortar, mixed with 10 mL of sterilized Ringer's solution, and shaken at 100 rpm for 30 min on an electrothermal constant-temperature shaking water tank. Sonicate in an ultrasonic apparatus for 1 min. Dilute the mixture appropriately and count using a hemocytometer 12 .
Determination of Leakage Count of immobilized yeast: 1 g of immobilized yeast was mixed with 10 mL of sterilized Ringer's solution and shaken at 100 rpm for 30 min on an electrothermal constant temperature shaking water tank. The initial concentration of the mixture was counted using a hemocytometer 12 . Comparison of between immobilized yeast and free yeast. The optimal red raspberry wine brewing process is the temperature of 26 °C, the sugar content of 220 g/L, and the immobilized yeast inoculum of 1.2%. Under these conditions, immobilized yeast and free yeast were fermented respectively, and the raspberry wines brewed by different fermentation methods were compared and analyzed in terms of changes in residual sugar, alcohol content, aroma components, and sensory evaluation.
Afterwards, the immobilized yeast was fermented for three times, and the repeated fermentation performance of the immobilized yeast was evaluated from the changes of residual sugar and alcohol content.

Detection of red raspberry wine. Alcohol test:
The alcohol content test refers to GB5009.225-2016 "National Food Safety Standard Determination of Ethanol Concentration in Wine". Aroma component detection: 1 g NaCl was placed in a 20 ml headspace vial, and the cap was tightened. After equilibrating at 60 °C for 5 min in stirring mode, headspace extraction was performed at 60 °C for 20 min with a solid-phase microextraction needle, and then desorbed at the injection port for 5 min. The chromatographic column is HP-INNOWAX capillary column (30 m × 0.25 mm × 0.25 μm); the carrier gas is He, the flow rate is 1 mL/min, the separation ratio is 5:1; the injection temperature is 250 °C; the heating program is the initial temperature of 40 °C, hold for 5 min, increase to 250 °C at 8 °C/min, hold for 5 min.Mass spectrometry conditions: EI ionization source, energy 70 eV; ion source temperature 230 °C, quadrupole temperature 150 °C, interface temperature 250 °C, scanning range 30-400 m/z.
Charting. All charts in this article are drawn by origin, or drawn by the test instrument independently.

Results and discussion
Optimization of preparation conditions for immobilized yeast. Carbon source optimization. The type of carbon source added has an effect on the size of the immobilized yeast and the clarity of the medium ( Table 3). The diameters of the immobilized yeast from large to small were lactose, glycerol, gluconic acid, glucose, and sucrose. At the same time, the larger the diameter, the smaller the number of immobilized yeast. The carbon source had no obvious effect on the surface state of the immobilized yeast, and the surface of the immobilized yeast with different carbon sources is smooth. However, different carbon sources had an effect on the clarity of immobilized yeast culture medium. When using glucose, sucrose, and glycerol, the medium is cloudy because the carbon source used is suitable for yeast growth 11 . Simultaneously yeast overgrowth causes the medium to be cloudy and inhibits the growth of P. chrysogenum. When lactose is used, the medium is yellow, while when gluconic acid is used, the medium is clear and normal. Therefore, considering the growth state of immobilized yeast, gluconic acid is determined as the best carbon source.
Optimization of nitrogen source. The type of nitrogen source added affects the size of the immobilized yeast and the surface state of the immobilized yeast ( Table 4). The diameters from large to small are urea, peptone, yeast extract, (NH4)2SO4, KNO3. Consistent with the effect of adding different carbon sources on immobilized yeast, the larger the diameter, the less the number of immobilized yeast. The immobilized yeast surface was rough when urea and peptone were used, indicating that nitrogen sources could affect the surface state of immobilized yeast. Based on the diameter and surface state of immobilized yeast, yeast extract was determined as the best nitrogen source.
Optimum dosage of Penicillium chrysogenum spores. The mycelium of P. chrysogenum, as the carrier of immobilized yeast, has a direct impact on the preparation of immobilized yeast 12 . The size of the immobilized yeast is different due to the different amount of P. chrysogenum spores added are 6 × 10 4 cfu/ml > 1 × 10 5 cfu/ml > 6 × 10 5 cfu /ml > 1 × 10 6 cfu/ml > 6 × 10 6 cfu/ml ( Table 5). The addition amount of P. chrysogenum can affect the diameter and number of immobilized yeast 13 . In terms of the number of immobilized yeasts, the more P. chrysogenum spores are added, the more immobilized yeasts will be. When the amount of spores added is large, more spores can interact with the yeast in a unit volume and can interact quickly, so it is easier and faster to form more immobilized yeasts with small diameters. On the other hand, because the number of yeasts is basically constant, the more spores of P. chrysogenum are added, the less yeast can be effectively encapsulated by each immobilized   www.nature.com/scientificreports/ yeast, and the volume is small. The medium with each concentration of P. chrysogenum spores was in a clear state and the surface was smooth, indicating that the addition of P. chrysogenum spores had no effect on the clarity of the medium, that is, the surface state of the immobilized yeast. Considering the quantity and size of immobilized yeast, the optimal spore addition range is 1 × 10 5 cfu/ml ~ 6 × 10 5 cfu/ml.
Orthogonal experiment. Based on the above, it is determined that the optimal carbon source is gluconic acid, the optimal nitrogen source is yeast extract, and the optimal addition amount of P. chrysogenum spores is 1 × 10 5 cfu/ml ~ 6 × 10 5 cfu/ml. Orthogonal optimization of the preparation conditions of immobilized yeast based on the optimal carbon source, nitrogen source and the addition amount of P. chrysogenum spores determined by single factor, design three-factor three-level orthogonal test of factors, the test level is shown in Table 6. The factors affecting the number of encapsulated bacteria in immobilized yeast were B > A > C, and the factors affecting the number of leaking bacteria in immobilized yeast were C > A > B. According to the orthogonal results (Table 7), when the diameter is used as the selection standard, the optimal combination is A2B2C1, namely No. 5. When the number of encapsulated bacteria is used as the selection criterion, the optimal combination is A2B1C3, namely No. 4. When the number of leaking bacteria was used as the selection criterion, the optimal combination was A2B2C1, namely No. 5.
Effects of mechanical strength of immobilized yeast. Immobilized yeast will be subjected to different mechanical forces during the fermentation process, resulting in crushing damage or scraping damage 14 , so the mechanical strength of immobilized yeast has an important impact on the performance and repeatability of immobilized yeast. The two groups of optimal combinations obtained from the optimized orthogonal test of immobilized yeast, the test groups No. 4 and No. 5, were tested for mechanical strength (Fig. 1a,b). The comparison between the two was shown in Fig. 1c. It can be seen that the hardness of No. 4 is 0.105 N and the elasticity is 0.135 mm.  Therefore, the optimal immobilized yeast was determined as the No. 5 test group, with 5 g/L of gluconic acid, 5 g/L of yeast extract, and 1 × 10 5 cfu/ml of P. chrysogenum spores.

Comparison between immobilized yeast and free yeast. Comparison of fermentation perfor-
mance. The results of residual sugar and alcohol content of red raspberry wine brewed by immobilized yeast and free yeast are shown in Fig. 2a,b. It can be seen that the residual sugar content of free yeast is slightly higher than that of immobilized yeast, and the sugar consumption rate of immobilized yeast is higher than that of free yeast, indicating that the fermentation rate of immobilized yeast is higher than that of free yeast.
The alcohol content of red raspberry wine brewed by immobilized yeast is 15% vol, and it brewed by free yeast is 14% vol. It was also proved that with the same inoculum amount, the fermentation ability of immobilized yeast was better than that of free yeast.
Comparison of ingredients in brewing red raspberry wine. The total acid, anthocyanin, reducing sugar and polyphenol content of red raspberry wine brewed with immobilized yeast and free yeast were analyzed.
It can be seen that the total acid content of the red raspberry wine brewed by immobilized yeast is 1.0438%, which is lower than 1.3687% of the red raspberry wine brewed by free yeast (Fig. 3a). That is, the acidity of red raspberry wine brewed by immobilized yeast is lower than that of free yeast, and the taste of red raspberry wine brewed by immobilized yeast is also better than that of free yeast.
The anthocyanin content of immobilized yeast brewed red raspberry wine was 111.604 mg/L, the reducing sugar content was 23.73 g/L, and the polyphenol content was 565.67 mg/L; the red raspberry wine brewed by www.nature.com/scientificreports/ free yeast was 68.020 mg /L, 21.93 g/L, 395 mg/L respectively (Fig. 3b,c,d).The reducing sugar content of red raspberry wine brewed by free yeast and immobilized yeast has little difference, but the content of anthocyanins and polyphenols in immobilized yeast is higher. It shows that immobilized yeast can better retain the active components of red raspberry wine compared to free yeast.
Comparison of aroma components in brewing red raspberry wine. Figures 4 and 5 for the GC-MS total ion efflux of aroma components in immobilized yeast and free yeast fermented red raspberry wine. A total of 40 main aroma components were detected in the two red raspberry wines, including 5 alcohols, 21 lipids, 3 acids, 3 alkenes, 3 aldehydes and ketones, 1 phenol, and 4 others. The two red raspberry wines have the same 22 kinds of aroma components, mainly alcohols and esters. It can be seen from Table 8 that the main aroma components of the red raspberry wine brewed by immobilized yeast from high to low are isoamyl alcohol 6.8312%, isobutanol 2.8885%, ethyl acetate 2.7084%, isoamyl acetate 2.5946%, ethyl caprylate 2.0175%. The main aroma components of red raspberry wine brewed with free yeast from high to low are isoamyl alcohol 6.0499%, ethyl acetate 2.8018%, isobutanol 2.7780%, isoamyl acetate 2.0802%, ethyl caprylate 1.3425%.
The most abundant aroma component in both raspberry wines was isoamyl alcohol. The total alcohol content of red raspberry wine brewed by immobilized yeast was 11.3627%, which was higher than that of free yeast brewed red raspberry wine 10.6472%. The lipid content of the red raspberry wine brewed with immobilized yeast was 10.0166%, which was higher than that of the red raspberry wine brewed with free yeast, which was 8.6791%. Compared with free yeast fermentation (Fig. 6), immobilized yeast can significantly increase the ester content 15 . Other studies have also shown that the use of immobilized yeast for fermentation can promote the production of aroma compounds such as alcohols and esters 9 . In addition, the immobilized yeast also effectively reduced the content of volatile acids, which was beneficial to the quality maintenance of red raspberry wine 16 .    Figure 7a,b show the changes of residual sugar content and alcohol content of immobilized yeast for three consecutive fermentations. From the results, the residual sugar content of three consecutive fermentations was 9.5%, 10%, and 10.5%, respectively. Alcohol content the degrees were 15% vol, 14.5% vol, and 14.2% vol. With the increase of fermentation times, the residual sugar content increased slightly, and the alcohol content decreased slightly, but the changes were not obvious, indicating that the immobilized yeast of P. chrysogenum maintained good fermentation performance after three consecutive fermentations.

Conclusion
The immobilized yeast of P. chrysogenum can be prepared by adding P. chrysogenum spores and co-culturing with yeast in a shaker flask. The optimal preparation conditions were determined by orthogonal optimization as 5 g/L of gluconic acid, 5 g/L of yeast extract, and 1 × 105 cfu/ml of P. chrysogenum spores. The obtained immobilized yeast has good physiological characteristics and mechanical properties.
In the fermentation process of red raspberry wine, the immobilized yeast of P. chrysogenum has better fermentation power than free yeast, and can better retain the active components such as anthocyanins and polyphenols in red raspberry wine, and at the same time obviously. The content of aroma components such as alcohols and lipids in red raspberry wine was improved. The immobilized yeast of P. chrysogenum maintained good fermentation performance after three consecutive fermentations.

Data availability
All data generated or analysed during this study are included in this published article. RelaƟve content /% Immobilized yeast Free yeast Figure 6. Aroma composition of red raspberry wine fermented with Immobilized yeast or free yeast.